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Towards Gene Therapy for CTLA4 Insufficiency

Fox, Thomas Andrew; (2022) Towards Gene Therapy for CTLA4 Insufficiency. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Heterozygous mutations in CTLA4 result in an inborn error of immunity (IEI) with a severe clinical phenotype. Autologous T cell gene therapy may offer a cure without the immunological complications of allogeneic stem cell transplantation. The mutational landscape and requirement for tight regulation of CTLA4 make viral gene addition approaches unappealing. Gene editing strategies permit alteration of CTLA4 while retaining the endogenous gene control machinery. We set out to devise a CRISPR/Cas9/AAV6 gene editing strategy to correct CTLA4 insufficiency in T cells. We designed several homology directed repair (HDR) editing strategies that would correct the genetic defect. We first assessed correction of an individual point mutation. We then evaluated several universal strategies that enable correction of most disease-causing mutations with a single edit; the first that inserts the CTLA4 cDNA in exon 1, and a second that inserts the CTLA4 cDNA at the 3’end of the first intron of CTLA4. Superior editing efficiencies were obtained with the intronic approach compared to the other editing strategies and this strategy was then further evaluated. CTLA4 function and expression kinetics were assessed following editing using flow cytometry-based assays. Functional studies using CTLA4 transendocytosis (TE) assays, demonstrated restoration of CD80 and CD86 internalization in the edited CD4+ T cells. Following gene editing, transgene expression kinetics were comparable to healthy control CD4+ T cells. Gene editing of T cells isolated from patients with CTLA4 insufficiency restored CTLA4 expression and rescued transendocytosis of CD80 and CD86 in vitro. Using a similar approach, gene corrected T cells from CTLA4 -/- mice engrafted in immunodeficient mice at clinically relevant frequencies and prevented lymphoproliferation in an in vivo murine model of CTLA4 insufficiency. These results demonstrate the feasibility of a novel therapeutic approach using T cell gene therapy for CTLA4 insufficiency.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Towards Gene Therapy for CTLA4 Insufficiency
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2021. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
Keywords: Gene editing, Primary immunodeficiency
UCL classification: UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Infection and Immunity
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10146895
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